Summary HIV-associated neurocognitive disorders (HANDs) and substance abuse comorbidity remain prevalent despite combination antiretroviral therapy (cART). A major challenge in the AIDS therapeutic development field is the identification of targetable mechanisms that underlie persistent neuronal dysfunction in HIV-infected individuals despite ART. While internal RNA modifications have been known for decades, their roles in RNA metabolism and function are only beginning to be elucidated. In particular, dynamic RNA modifications are believed to represent a new layer of control of gene expression. The present proposal will test the overarching hypothesis that understanding the role of RNA modifications in the implementation of gene expression programs that are dysregulated by the interaction of HIV, cART and substance abuse in neurons, astrocytes and microglia, can indicate transformative new mechanistic hypotheses on neuroAIDS pathogenesis that will have the potential to lead to the identification of novel therapeutic targets to improve neuropsychological functioning in people with HIV. The impact of RNA modifications on HIV transcripts and splicing and HIV protein levels in HIV Tg rats will also be investigated. To test the present hypothesis we assembled a collaborative team involving expertise in behavioral and molecular neurobiology, gene expression and advanced systems biology methods to study transcriptional, post-transcriptional and post-translational regulatory mechanisms. In particular, we will explore RNA modifications in well-established behavioral paradigms of moderate or compulsive (dependent) cocaine self-administration in HIV transgenic (Tg) rats in the setting of combination antiretroviral therapy (cART). We will use a systems biology approach in conjunction with RNA profiling in identified brain cell types to investigate the role of RNA modifications in neuronal injury and glia dysfunction in neuroAIDS. We will focus primarily on the most abundant mRNA modification, N6-methyladenosine (m6A) and the proteins that compose its regulatory machinery. The effects of histories of cocaine intake and cART on m6A modification of HIV transcripts in HIV Tg rats will also be investigated. The mechanisms identified will also be investigated in follow-up studies in models of moderate and compulsive self-administration of other drugs of abuse as well as available human gene expression datasets. Ultimately this proposal - at the interface of computational analysis of gene network regulation and advanced behavioral pharmacology - is aimed at identifying new testable mechanistic hypotheses that may lead to transformative new therapeutic concepts to improve neuropsychological functioning in people with HIV.